Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation

ABSTRACT

Disclosed are methods for producing fusion proteins with the heterodimeric cytokine, interleukin-12. In order to insure that the proper ratio of fused and non-fused subunits are obtained in the fusion protein, a specific stepwise approach to genetic engineering is used. This consists of first expressing the non-fused p40 IL-12 subunit in a production cell line, followed by or simultaneously expressing in the same cell, a second recombinant fusion protein consisting of the fused polypeptide linked by a peptide bond to the p35 subunit of IL-12. Molecules containing the p35 fusion protein cannot be secreted from the transfected mammalian cell without first complexing in a one to one ratio with the p40 subunit, thus ensuring the production of active heterodimeric fusion proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/986,997, filed onDec. 8, 1997, now abandoned the disclosure of which is incorporated byreference in its entirety herein.

FIELD OF THE INVENTION

The present invention relates generally to fusion proteins. Morespecifically, the present invention relates to heterodimeric fusionproteins useful for targeted immune therapy and general immunestimulation.

BACKGROUND OF THE INVENTION

One of the key immune regulators is the T helper cell which reacts toantigens presented on HLA class II molecules. This CD4⁺ celldifferentiates in response to antigenic stimulation and becomes a type 1or type 2 helper (Th1 or Th2) according to the type of cytokines that itsecretes. Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173 (1989). ATh1 response leads to the secretion of interleukin-2 (IL-2) andinterferon-γ (IFN-γ) which stimulates cell-mediated immune reactionsagainst intracellular pathogens. A Th2 response leads to the secretionof IL-4, IL-5 and IL-10 which stimulates antibody responses toextracellular pathogens. The most interesting component of this systemof regulation is that one response inhibits the other through thenegative regulatory activities of the cytokines that are produced. Thus,IL-4 and IL-10 can down-regulate Th1 responses while IFN-γ candown-regulate Th2 responses.

The regulatory activity of T helper cells and their differentiationfollowing exposure to antigen is regulated by cytokines as well. IL-12,a disulfide-linked heterodimeric cytokine with a 40 kDa subunit and 35kDa subunit, exerts a powerful positive regulatory influence on thedevelopment of Th1 helper T-cell immune responses. See review byTrinchieri, Blood 84: 4008-4027 (1994). IL-12 also has a powerfulsynergistic effect in the induction of IFN-γ from both T helpers andnatural killer (NK) cells (Eur. Patent Appl. 90123670.3). Secreted IFN-γthen inhibits any Th2 cell proliferation and polarizes the response tofavor cell-mediated immunity.

One way of changing the outcome of an immune response would be toadminister the appropriate cytokine at the time of antigen stimulation.If IL-4 was the major cytokine present during antigen stimulation, theTh2 response would be enhanced and the Th1 response would be inhibited.In contrast, if IL-12 was the major cytokine present during antigenstimulation, the Th1 response would be enhanced and the Th2 responsewould be inhibited. However, systemic administration of cytokines isdifficult due to their very short circulating half-lives and theirdeleterious side effects.

A better approach is to target the effect of the cytokine to a cellsurface antigen by fusing it to an antibody (or fragment derivedtherefrom) having specificity and affinity for that antigen. SeeGillies, et al., Proc. Natl. Acad. Sci. 89: 1428-1432 (1992); U.S. Pat.No. 5,650,150, the disclosure of which is incorporated herein byreference. Alternatively, the stimulatory cytokine can be linked to aprotein antigen via a peptide linkage in the form of a fusion protein.See Hazama, et al., Vaccine 11: 629-636 (1993). However, the complexstructure of IL-12 makes it more difficult to express as a fusionprotein due to the necessity of expressing exactly the same molar ratioof each subunit in the final product. In fact, IL-12 itself is naturallyexpressed and secreted as a mixture of p40 homodimer. D'Andrea, et al.,J. Exp. Med., 176:1387-1398 (1992).

Therefore, there is a need in the art for methods of producing fusionproteins with heterodimeric cytokines and an antibody or an antigen thatmaintain the natural heterodimeric structure of the cytokine andsecretes the molecules with equimolar ratios of the subunits.

SUMMARY OF THE INVENTION

The present invention provides heterodimeric fusion proteins useful fortargeted immune therapy and general immune stimulation and methods forproducing these heterodimeric fusion proteins. Specifically, the presentinvention provides methods for the production of fusion proteins withIL-12 that maintain its natural heterodimeric structure, and provide forthe secretion of the molecules with equimolar ratios of IL-12 subunits.

In one aspect of the invention, the fusion proteins comprise aheterodimeric cytokine linked to an antibody, or a portion thereof. In apreferred embodiment, the fusion protein comprises two chimeric chainslinked by a disulfide bond. Each chimeric chain comprises a differentsubunit of the heterodimeric cytokine linked through a peptide bond to aportion of an Ig heavy chain.

In an alternative preferred embodiment, the fusion protein comprises afirst chimeric chain comprising one of the subunits of the heterodimericcytokine linked by a peptide bond to a portion of an Ig heavy chain.This subunit is linked by a disulfide bond to the other subunit of theheterodimeric cytokine. In another alternative preferred embodiment,this first chimeric chain is linked by a disulfide bond to a secondchimeric chain comprising one of the subunits of the heterodimericcytokine linked by a peptide bond to a portion of an Ig heavy chain andby a disulfide bond to the other subunit of the heterodimeric cytokine.

In yet another alternative preferred embodiment, the fusion protein is atrimeric fusion protein comprising a first and a second chimeric chainlinked by a disulfide bond. Each chimeric chain comprises a subunit ofthe heterodimeric cytokine linked by a peptide bond to a portion of anIg heavy chain. The subunit of one of the chimeric chains is furtherlinked by a disulfide bond to a different subunit of the heterodimericcytokine.

Fusion proteins of the invention may be considered chimeric by virtue oftwo aspects of their structure. First, the fusion protein is chimeric inthat it includes an immunoglobulin chain (typically but not exclusivelya heavy chain) of appropriate antigen-binding specificity fused to agiven heterodimeric cytokine. Second, an immunoconjugate of theinvention may be chimeric in the sense that it includes a variableregion and a constant region which may be the constant region normallyassociated with the variable region, or a different one and thus a V/Cchimera; e.g., variable and constant regions from different species.Also embraced within the term “fusion protein” are constructs having abinding domain comprising framework regions and variable regions (i.e.,complementarity determining regions) from different species, such as aredisclosed by Winter, et al., GB2, 188, 638.

The heterodimeric cytokine-antibody fusion protein of the presentinvention preferably displays antigen-binding specificity. In apreferred embodiment, the heterodimeric cytokine-antibody fusion proteincomprises a heavy chain. The heavy chain can include a CH1, CH2, and/orCH3 domains. In an alternative preferred embodiment, the heterodimericcytokine-antibody fusion protein comprises a light chain. The inventionthus provides fusion proteins in which the antigen binding specificityand activity of an antibody are combined with the potent biologicalactivity of a heterodimeric cytokine. A fusion protein of the presentinvention can be used to deliver selectively a heterodimeric cytokine toa target cell in vivo so that the heterodimeric cytokine can exert alocalized biological effect.

Preferably, the fusion protein of the present invention displayscytokine biological activity. The preferred heterodimeric cytokine ofthe fusion protein is IL-12. Fusions with antibodies capable of bindingantigens are useful for co-localizing the immune stimulatory activity ofIL-12 either to target cells or target protein antigens.

Further, the fusion protein of the present invention preferably has alonger circulating half-life than an unlinked heterodimeric cytokine.Fusions with the Fc portion of antibodies and IL-12 are useful foraltering the pharmacology and biodistribution of the molecule byincreasing its circulating half-life and its affinity for Fc-receptorbearing cells, e.g., antigen presenting cells. Changes inbiodistribution may also alter its systemic toxicity by changing themechanism by which it is cleared from the circulation.

In another aspect of the invention, the fusion proteins comprise aheterodimeric cytokine linked to an antigen. The preferred heterodimericcytokine-antigen fusion protein of the present invention displayscytokine biological activity and antigenic activity. Further, the fusionprotein of the present invention preferably has a longer circulatinghalf-life than an unlinked heterodimeric cytokine. The preferredheterodimeric cytokine of the fusion protein is IL-12.

In a preferred embodiment, the fusion protein comprises two chimericchains linked by a disulfide bond. Each chimeric chain comprises adifferent subunit of the heterodimeric cytokine, either of which islinked through a peptide bond to an antigen.

In an alternative preferred embodiment, the fusion protein comprises afirst chimeric chain comprising one of the subunits of the heterodimericcytokine linked by a peptide bond to an antigen. This subunit is linkedby a disulfide bond to the other subunit of the heterodimeric cytokine.In another alternative preferred embodiment, this first chimeric chainis linked by a disulfide bond to a second chimeric chain comprising oneof the subunits of the heterodimeric cytokine linked by a peptide bondto an antigen and by a disulfide bond to the other subunit of theheterodimeric cytokine.

In another alternative preferred embodiment, the fusion protein is atrimeric fusion protein comprising a first and a second chimeric chainlinked by a disulfide bond. Each chimeric chain comprises a subunit ofthe heterodimeric cytokine linked by a peptide bond to an antigen. Thesubunit of one of the chimeric chain is further linked by a disulfidebond to a different subunit of the heterodimeric cytokine.

The invention also features DNA constructs encoding the above-describedfusion proteins, and cell lines, e.g., myelomas, transfected with theseconstructs.

The invention also includes a method for selectively targeting aheterodimeric cytokine. In a preferred embodiment, the method compriselinking at least one subunit of a heterodimeric cytokine by a peptidebond to a portion of an Ig heavy chain. In an alternative preferredembodiment, the method comprise linking each of the two subunits of aheterodimeric cytokine by a peptide bond to a portion of an Ig heavychain, thereby forming two chimeric chain. The two chimeric chains arelinked by a disulfide bond, thereby forming a heterodimeric fusionprotein. In yet another preferred embodiment, the method comprises (1)linking one of the two subunits of a first heterodimeric cytokine by apeptide bond to an Ig heavy chain, thereby forming a first chimericchain; (2) linking one of the two subunits of a second heterodimericcytokine by a peptide bond to an Ig heavy chain, thereby forming asecond chimeric chain; and (3) linking the first and second chimericchains by a disulfide bond, thereby forming a fusion protein. Theresulting fusion proteins can display binding specificity for apredetermined antigen and cytokine biological activity.

The invention also includes a method of selectively delivering aheterodimeric cytokine to a target cell. The method includes providing aheterodimeric cytokine fusion protein including a chimeric Ig chainincluding an Ig heavy chain having a variable region specific for anepitope on the target cell and a constant region joined at its carboxyterminus by a peptide bond to a cytokine, and an Ig light chain combinedwith the chimeric Ig heavy chain, forming a functional antigen-bindingsite, and administering the fusion protein in an amount sufficient toreach the target cell to a subject harboring the target cell.

Further, the invention features a method of increasing the circulatinghalf-life of a heterodimeric cytokine. In a preferred embodiment, themethod comprise linking at least one subunit of a heterodimeric cytokineby a peptide bond to a polypeptide. In an alternative preferredembodiment, the method comprises linking each of the two subunits of aheterodimeric cytokine by a peptide bond to a polypeptide, therebyforming two chimeric chain. The two chimeric chains are linked by adisulfide bond, thereby forming a heterodimeric fusion protein. In yetanother preferred embodiment, the method comprises (1) linking one ofthe two subunits of a first heterodimeric cytokine by a peptide bond toa polypeptide, thereby forming a first chimeric chain; (2) linking oneof the two subunits of a second heterodimeric cytokine by a peptide bondto a polypeptide, thereby forming a second chimeric chain; and (3)linking the first and second chimeric by a disulfide bond, therebyforming a fusion protein. The polypeptide can be serum albumin, anantigen, and a portion of an Ig heavy chain. The resulting fusionproteins display cytokine biological activity.

The IL-12 fusion proteins of the present invention are useful forspecific targeting or immune stimulation when it is important togenerate a cell-mediated immune response, such as in cancerimmunotherapy or antiviral responses. They are also useful forspecifically downregulating Th2 responses which often lead to theoverproduction of IL-4. This cytokine has been shown to be essential forthe development of allergy through the induction of a Th2 response andthe resulting overproduction of IgE antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present invention, and thevarious features thereof, may be more fully understood from thefollowing description, when read together with the accompanyingdrawings, in which:

FIG. 1 is a diagrammatic representation of the predicted proteinstructure of heterodimeric fusion proteins;

FIG. 2 is a diagrammatic representation of an SDS-PAGE showing ananalysis, under reducing conditions, of proteins secreted by cellstransfected with vectors expressing the Fc-p35 fusion protein (lane 1),the Fc-p40 fusion protein (lane 2), the Fc-p35 fusion protein and theFc-p40 fusion protein (lane 3), the Fc-p35 fusion protein and the p40subunit (lane 4), and the p35 subunit and the Fc-p40 fusion protein(lane 5);

FIG. 3 is a diagrammatic representation of the predicted proteinstructure of expressed fusion proteins;

FIG. 4 is a bar graph depicting the ability of various fusion proteinsto stimulate IFN-γ production;

FIG. 5A is a diagrammatic representation of an SDS-PAGE showing ananalysis of whole antibody-IL-12 fusion proteins produced by twoindependent transfectants, under non-reducing (lanes 1 and 2) andreducing conditions (lanes 3 and 4);

FIGS. 5B-D are line graphs depicting the effects of human IL-12 (X),Hu-KS-IL-12 fusion protein with both human IL-12 chains (closedsquares), and Hu-KS-¼-mouse p35 human p40 fusion protein (open squares)on proliferation of mitogen-activated human PBMC (Panel B); induction ofIFN-γ secretion from PHA-activated PBMC (Panel C) and from mouseeffector cells, pre-stimulated with Concanavalin A (Panel D);

FIGS. 6A-B are line graphs depicting the effects of IL-12 (X),single-chain fusion protein with human p35 and p40 subunits (closedsquares), and single-chain fusion protein with a mouse p35 subunit and ahuman p40 subunit (open squares) on induction of IFN-γ secretion;

FIG. 6C is line graphs depicting the antigen binding activity of wholeHu-KS-¼-IL-12 fusion protein (open squares), single-chain fusion proteinwith human IL-12 (open diamond), single-chain fusion protein with mousep35 human p40 (open and free circles), and human IL-12 (open triangles);

FIG. 7 is a graph depicting the serum half-life of Hu-KS-IL-12 (mousep35 human p40), as measured by an ELISA using a capture step withanti-human H and L chain and a second detection with either anti-humanFc antibody (closed diamonds) or anti-human IL-12 p40 antibody (opensquares);

FIG. 8 (top and bottom panels) are line graphs depicting theimmunogenicity of IL-12 fusion proteins. Serum dilutions from animalsinjected with either Hu-KS-¼ antibody or Hu-KS-¼-IL-12 (mouse p353 humanp40) were tested for reactivity to Hu-KS-¼ antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes fusion proteins between heterodimericcytokines and other proteins. Heterodimeric cytokines can be fused to,for example, proteins with targeting or antigenic properties. Fusionproteins between heterodimeric cytokines and proteins with targeting orantigenic properties may have a longer circulating half life thanunlinked heterodimeric cytokines. Targeting or antigenic properties arenot required for the increased circulating half life as this propertycan also be achieved by fusing a heterodimeric cytokine with a proteinthat lacks targeting or antigenic properties such as, for example, serumalbumin.

The fusion proteins of this invention can be produced by geneticengineering techniques. As depicted in FIG. 1, various fusion proteinconstructs can be produced by the methods of the present invention. Inone embodiment, one of the subunit of the heterodimeric cytokine fusedto a polypeptide is co-expressed with a free subunit of the other type.Once expressed, the chimeric chain is linked by a disulfide bond to thefree subunit (FIG. 1B). In another embodiment, the polypeptide fusedwith one of the subunit can be linked to another such polypeptide. Sinceeach polypeptide is linked to a heterodimeric cytokine, the resultingconstruct has two molecules of the heterodimeric cytokine (FIG. 1C). Inyet another embodiment, each of the subunit of the heterodimericcytokine is fused to a polypeptide and the two chimeric chains arelinked by a disulfide bond. The resulting construct has only onemolecule of the heterodimeric cytokine (FIG. 1D). In yet anotherembodiment, two subunits of the heterodimeric cytokine fused to apolypeptide are co-expressed with a free subunit. The resultingconstruct has three subunits of the heterodimeric cytokine (FIG. 1E).

At present, the only known heterodimeric cytokine is IL-12. However, asnovel heterodimeric cytokines are identified and sequenced, a skilledartisan will be able to use methods of the present invention to producefusion proteins with these novel heterodimeric cytokines.

Methods for synthesizing useful embodiments of the invention aredescribed, as well as assays useful for testing their pharmacologicalactivities, both in vitro and in pre-clinical in vivo animal models. Thepreferred gene construct encoding a chimeric chain (i.e., a subunit ofthe heterodimeric cytokine fused to a polypeptide) includes, in 5′ to 3′orientation, a DNA segment which encodes a polypeptide and DNA codingfor one subunit of the heterodimeric cytokine. An alternative preferredgene construct includes, in 5′ to 3′ orientation, a DNA segment whichencodes one subunit of the heterodimeric cytokine and DNA coding for apolypeptide. The fused gene is assembled in or inserted into anexpression vector for transfection of the appropriate recipient cellswhere it is expressed.

The invention is illustrated further by the following non-limitingexamples:

EXAMPLE 1 Cloning cDNAs Encoding Human and Mouse IL-12 Subunits

Human peripheral blood monocytes (PBMC) were obtained from a healthyvolunteer and were purified by centrifugation on a Ficoll-Hypaque(Pharmacia) gradient (1700 rpm for 20 min). The “buffy” coat containingthe PBMC was diluted with serum-free culture medium (SF-RPMI) to avolume of 50 ml and collected by centrifugation at 1500 rpm for 5 min.Cells were resuspended in AIM-V cell culture medium (GIBCO) at a densityof 5×10⁶ cells/ml and were cultured for 2 days at 37° C. in a humidifiedCO₂ incubator. The attached cells were selected by gently agitating theculture flask to remove non-adherent cells. Fresh medium containingphorbol ester (100 nM) and the calcium ionophore, ionomycin (0.1 μg/ml)was added. After three days, the cells were collected by gentle scrapingand centrifugation. Poly A+mRNA was prepared using oligo dT-coated beads(Dynal, Inc.).

Subunit cDNAs were cloned using polymerase chain reactions (PCR). Firststrand cDNA was synthesized in a 50 μl reaction containing oligo dTprimer (50 μg/ml), reaction buffer, RNAsin (10 U/ml) and reversetranscriptase. Incubation was at 43° C. for 2 hrs, followed byextraction with phenol, phenol:chloroform (50:50) and precipitation withethanol. The cDNA product was used as template for PCR reactionscontaining Taq polymerase and reaction buffer (10×buffer; Perkin Elmer),sense and antisense primers (0.2 to 0.5 μM each), and 10% of the cDNAreaction. Primer sequences were 5′-CCAGAAAGCAAGAGACCAGAG-3′ (SEQ IDNO: 1) for the sense primer, and 5′-GGAGGGACCTCGAGTTTTAGGAAGCATTCAG-3′(SEQ ID NO: 2) for the antisense primer of the p35 subunit cDNA. Thesense primer is derived from a sequence in the 5′ untranslated region ofthe p35 message just upstream of a XmaI site, while the antisense primerencodes a translational stop codon followed shortly thereafter by aconvenient XhoI site for directional subcloning in an expression vector.The primers for the p40 subunit cDNA were5′-CTCCGTCCTGTCTAGAGCAAGATGTGTC-3′ (SEQ ID NO: 3) for the sense and5′-GCTTCTCGAGAACCTAACTGCAGGGCACAG-3′ (SEQ ID NO: 4) for the antisenseprimer. The sense primer encodes a unique XbaI site upstream of thetranslation start site while the antisense primer encodes a stop codonand unique XhoI site as above. Both subunit sequences, cloned with thesePCR primers, will be expressed as single proteins and thus requirenative (or other) secretory leader sequences for proper heterodimerassembly and secretion. PCR reactions consisted of 40 cycles including:1 min at 92° C., 2 min at 52° C, and 3 min at 72° C., following aninitial denaturation step at 94° C. for 2 min. Products were gelpurified and cloned in the SK cloning vector (Strategene) for sequenceverification. DNA sequencing using a commercial kit (U.S. Biochemical)was carried out on each of the subunit cDNA. The same procedure can beused to clone the mouse p35 subunit cDNA from spleen cells activatedwith Concanavalin A (5 μg/ml in culture medium for 3 days). Recommendedprimers are 5′-CCTCTACTAACATGTGTCAATCACGCTACCTC-3′ (SEQ ID NO: 5) forthe sense and 5′-CCCTCGAGTCAGGCGGAGCTCAGATAGCC-3′ (SEQ ID NO: 6) for theantisense primers encoding the same restriction sites as described abovefor the human p35 subunit.

EXAMPLE 2 Expression of Fusion Protein Combinations in TransfectedMammalian Cells

In order to make the fused versions of each subunit, the DNAs encodingthe mature protein sequence of each were adapted as follows. The p40subunit DNA was digested with NdeI which cuts very close to the junctionof the mature protein and leader sequence, and XhoI. An adapteroligonucleotide was synthesized with the sequence 5′-CCGGGCAAGTCCA-3′(SEQ ID NO: 7) hybridized to a second, partly complementaryoligonucleotide with the sequence 5′-TATGGACTTGC-3′ (SEQ ID NO: 8). Thedouble stranded DNA contains overhanging sequence compatible withligation to an XmaI site at the 5′ end and an NdeI site at the 3′ end.This fragment was ligated to the NdeI-XhoI fragment of the p40 cDNA andcloned as an XmaI to XhoI fragment in vector pdC-Fc-X, cut with XmaI andXhoI. This vector already contains a human IgGI Fc encoding DNA fragmentin its genomic configuration (containing introns and exons) and fuseddownstream of a leader sequence derived from a mouse light chain. See,Gillies, et al., J. Immunol. Methods 125: 191-202 (1989). The additionof a DNA fragment to its unique XmaI site allows for the production offusion proteins joined directly to the carboxyl terminus of the Fc,provided that the reading frame between the two sequences is maintained(Lo, et al., U.S. Pat. No. 5,541,087). Other proteins (e.g., antigen,serum albumin) can be fused to the amino termini of these subunits inthe same manner. The advantages of this method include the largequantities of product produced and the ease of purification of theproduct by binding to and elution from protein A Sepharose.

The same general strategy was used to fuse the p35 subunit DNA to humanFc. In this case, a XmaI-BalI linker was synthesized using theoligonucleotides 5′-CCGGGAAGAAACCTCCCCGTGG-3′ (SEQ ID NO: 9) and5′-CCACGGGGAGGTTTCTTC-3′ (SEQ ID NO: 10), which were ligated to a p35subunit DNA, cut with BalI and XhoI, and subcloned as an XmaI-XhoIfragment in the pdC-Fc-X vector, as described above. The human p35subunit has been shown to be active for human cells but not mouse cells,in terms of IL-12 activity, whereas the human p40 subunit does not showspecies specificity. Therefore, the human p40 subunit can be used tomake either all human IL-12 fusion proteins or hybrid human/mouse fusionproteins.

The resulting constructs encode Fc-p35 or Fc-p40 fusion proteins whichare expected to spontaneously dimerize into proteins of 120 kD (50 Kdfrom the Fc) and 130 kD respectively and to migrate after reduction ondenaturing SDS gels as proteins of 60 kD and 65 kD. The individualsubunit cDNAs were subcloned in the pdC expression vector (without theFc) for their expression as independent proteins. This vector providespromoter sequences for expression of mRNA, transcribed from the cDNAinsert, following the transfection of mammalian cells. It also providesfor a 3′ untranslated region and poly A addition site, downstream of the3′ XhoI insertion site. There are also sequences necessary for thepropagation of the plasmid in E. coli and selection with ampicillin, aswell as a selectable marker gene, such as dihydrofolate reductase(dhfr), for conferring resistance to methotrexate. These same componentsare also used in the pdC-Fc-X vector for expression of the fusionproteins.

For expression of biologically-active IL-12 fusion protein heterodimers,different combinations of the individual vectors encoding fusion andnon-fusion forms of the subunits were transiently expressed byco-transfection of human 293 epidermal carcinoma cells. DNA was purifiedusing preparative kits (Wizard, Promega Inc.), ethanol precipitated forsterilization and resuspension in sterile water. Calcium phosphateprecipitates were prepared by standard methods using 10 μg of DNA per ml(5 μg of each when two plasmids were co-transfected) and 0.5 ml/platewere added to cultures of 293 growing in 60 mm plates at approximately70% confluency. MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed.(Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor LaboratoryPress, 1989). After 16 hr, the medium containing the precipitate wasremoved and replaced with fresh medium. After 3 days, the supernatantwas removed and analyzed for production of transfected gene expressionby ELISA, biological determination of IL-12 activity, orimmunoprecipitation and analysis on SDS gels of radioactively labeledproteins. For labeling, medium without methionine was used to replacethe growth medium on the second day of culture and ³⁵S-methionine (100μCi/ml) is added. After an additional 16 hr incubation, the media washarvested, clarified by centrifugation (5 min at 13,000 rpm in a tabletop microcentrifuge) and incubated with protein A Sepharose beads (10 μlof bead volume per ml of culture supernatant). After 1 hr at roomtemperature, the beads were washed by repeated centrifugation andresuspension in PBS buffer containing 1% NP-40. The final pellet wasresuspended in SDS-containing gel buffer and boiled for 2 min. Afterremoving the beads by centrifugation, the supernatant was divided intotwo aliquots. Reducing agent (5% 2-mercaptoethanol) was added to onesample and both are boiled for 5 min prior to loading on an SDSpolyacrylamide gel. After electrophoresis the gel was exposed to X-rayfilm (autoradiography).

An example of an analysis of the co-expression of various fusionproteins and individually expressed proteins, under reducing conditions,is shown in FIG. 2. The results show that the p35 subunit cannot besecreted from the cell, even when expressed as a fusion protein with theFc fragment (lane 1). The p40 subunit, on the other hand, was readilysecreted when fused to Fc (lane 2). The p35 subunit was secreted when itcould pair with the p40 subunit, either as an Fc-p35 fusion pairing withan Fc-p40 fusion protein (lane 3), the Fc-p35 pairing with free p40(lane 4), or free p35 pairing with the Fc-p40 fusion protein (lane 5).In all cases of expression of a free subunit, together with a fusionprotein, the free subunit assembles with the other subunit and forms acovalent, disulfide bond. A diagram of these various combinations isshown in FIG. 1. Note that the construct with each subunit fused to Fcand co-expressed in the same cell has one molecule of IL-12 per Fc (FIG.1D), whereas the constructs with a single subunit fusion to Fc pairedwith a free subunit (of the other type) has two molecules of IL-12 perFc (FIG. 1C). Expression in stably transfected cells is expected to bedifferent from transient expression since the expression and secretionis independent of p35. Thus, overexpression of p40 is possible and moreadvantageous to the cell since it can easily be exported. This couldlead to an overabundance of Fc-p40 subunits relative to Fc-p35 andresult in a mixture of heterodimer and p40 homodimer secretion from thecell. This would be inefficient and lead to purification problems.Expression of p35 is likely to have a growth disadvantage, since excessprotein is likely degraded in the endoplasmic reticulum, unless it iseffectively paired with the p40 subunit. Thus, it is possible to takeadvantage of this situation to ensure the balanced secretion of onlyheterodimer fusion product, by expressing the p35 subunit as a fusionprotein together with free p40 subunit. Only p35 fusion protein pairedwith an equimolar amount of p40 subunit can be secreted. Purification ofthis product on protein A results in a homogeneous preparation ofheterodimer. A diagrammatic representation of the predicted proteinstructure of expressed fusion proteins is provided in FIG. 3.

EXAMPLE 3 Activity of Fusion Proteins on in an IFN-γ Induction Assay

Biological activity was measured in an IFN-γ induction assay usingmitogen-activated human PBMC, purified as described in Example 1. Aftergradient centrifugation, cells were resuspended in cell culture mediumcontaining 10% fetal bovine serum (RPMI-10) and phytohemaglutinin (PHA;10 μg/ml) at a density of 5×10⁶ cells/ml and were cultured for 3 days at37° C. in a humidified CO₂ incubator. The PHA-activated cells werecollected by centrifugation, washed three times with an equal volume ofSF-RPMI and resuspended in fresh RPMI-10 (1×10⁶ cells /ml). Aliquots(100 μl) were dispensed into the wells of multiple 96-well plates togive a final cell number of 10⁵ per well. Test samples from culturemedium were serially diluted in fresh culture medium and added to wellsof the 96-well plate. Stimulation medium (50 μl/well) containing 10%serum and IL-2 (25 U/ml) was added. Control wells received only IL-2(negative control) or both IL-2 and commercial IL-12 (R & D Systems) butno sample (positive control). The plates were incubated for 48 hr at 37°C. in a CO₂ incubator at which time aliquots (20 μl) were removed foranalysis of IFN-γ concentration by ELISA.

The same assay was used to determine the activity of mouse forms ofIL-12 fusion proteins, except that spleen cells from Balb/c miceactivated for 3 days with Concanavalin A, were used instead ofPHA-activated human PBMC. A mouse-specific ELISA was used to quantitatethe amount of IFN-γ induced by the human p40/mouse p35 hybrid moleculesfrom mouse cells.

For the human system, a quantitative ELISA was developed by coating96-well plates (Nunc-Immuno plate F96 Cert. Maxisorb) with a mousemonoclonal antibody against human IFN-γ (1 μg/ml) in phosphate bufferedsaline (PBS; Pestka Biological Laboratories) overnight at 4° C., washingunbound antibody three times with PBS, and blocking with a solution of1% bovine serum albumin (BSA) and 1% goat serum in PBS (150 μl/well for2 hr at 37° C.). After washing the blocked plates four times with PBS,test samples and dilutions of the IFN-γ standard were added in a finalvolume of 100 μl/well. Following an overnight incubation at 4° C., theplates were washed four times with PBS, and a polyclonal rabbitantiserum against human IFN-γ ({fraction (1/10000)} dilution; PetskaBiological Laboratories) was added. After an additional incubation for 1hr at 37° C. and four washes with PBS, a polyclonal donkey anti-rabbitdetecting antibody, conjugated to horse radish peroxidase ({fraction(1/700)} dilution; Petska Biological Laboratories) was added for 1 hr at37° C. The plates are then washed four times with PBS and 100 μl ofK-blue substrate (ELISA Technologies, Neogen Corp.) was added until thecolor in the wells containing the standard curve was sufficientlydeveloped, at which time 100 μl of Red-stop solution (ELISATechnologies) was added. The plate was read at 650 nm using an ELISAplate reader (Dynatech MR7000) and the amount of IFN-γ was calculated bycomparing the optical density of the test sample with a standard curvederived from the dilutions of the control IFN-γ. The amount of IFN-γthat was induced in the presence of both IL-2 and IL-12 generally rangesfrom 1200-2000 pg/ml while the amount produced in the absence of IL-12was generally less than 50 pg/ml.

The biological activity of the culture supernatants described in Example2 were compared for their ability to stimulate IFN-γ production. Asdepicted in FIG. 4, the highest activity was obtained with the Fc-p35fusion protein co-expressed with free p40 subunit, although the othercombinations with both subunits were also active. More accuratemeasurements with purified proteins are described below.

EXAMPLE 4 Expression of Antibody-IL-12 Fusion Proteins

The experiments described in Example 2 demonstrate that a convenient wayto express fusion proteins with the IL-12 heterodimeric cytokine is toco-express a fused p35 subunit protein together with the free p40subunit in the same cell. This can be done by two approaches: the firstis achieved by co-transfecting the fusion protein vector and the p40expression vector simultaneously (i.e., simultaneous transfection); thesecond is to first transfect a cell with p40 alone and select for highlevel, stable secretors of this protein, and then use this cell as arecipient for transfection by the fusion protein expressing construct(i.e., sequential transfection). The latter method is particularlyuseful when the fusion protein is an antibody molecule with both a heavyand light chain that need to be assembled properly for correct assemblyand secretion. Theoretically, the fusion of p35 subunit could be to theheavy or light chain, but the preferred embodiment would be to thecarboxyl terminus of the heavy chain, where it can be more free tointeract with the IL-12 receptor on cells. It is also possible to fusethe p35 subunit via its carboxyl terminus to the amino terminus of theheavy or light chain. In this case, a leader sequence would be requiredfor p35 expression, since it would be at the amino terminus of thefusion protein, thus requiring its direction to the endoplasmicreticulum for assembly and secretion from the cell.

The nucleic acid construct can also include the endogenous promoter andenhancer for the variable region-encoding gene to regulate expression ofthe chimeric immunoglobulin chain. For example, the variable regionencoding genes can be obtained as DNA fragments comprising the leaderpeptide, the VJ gene [functionally rearranged variable (V) regions withjoining (J) segment] for the light chain or VDJ gene for heavy chain,and the endogenous promoter and enhancer for these genes. Alternatively,the gene coding for the variable region can be obtained apart fromendogenous regulatory elements and used in an expression vector whichprovides these elements.

Variable region genes can be obtained by standard DNA cloning proceduresfrom cells that produce the desired antibody. Screening of the genomiclibrary for a specific functionally rearranged variable region can beaccomplished with the use of appropriate DNA probes such as DNA segmentscontaining the J region DNA sequence and sequences downstream.Identification and confirmation of correct clones are then achieved byDNA sequencing of the cloned genes and comparison of the sequence to thecorresponding sequence of the full length, properly spliced mRNA.

4.1 Simultaneous Transfection

Simultaneous transfection can be achieved by constructing a vector withtwo transcription units and a selectable marker gene. Such vectors aredescribed for the expression of recombinant antibodies in mammaliancells. Gillies, et al., J. Immunol. Methods 125: 191-202 (1989). Analternative method is to use two independent plasmid vectors (one with atranscription unit for the fusion protein and one with a transcriptionunit for the p40 subunit) with their own selectable marker genes, and toselect for successfully transfected, expressing cells by culturing inthe presence of the drugs to which the cells have become resistant(e.g., methotrexate in cells transfected with the dhfr gene). Stillanother approach would be to use an expression vector for the fusionprotein to the p35 subunit containing a selectable marker gene andco-transfecting a second vector with no selectable marker gene and atranscription unit for the p40 subunit. Any drug resistant cloneobtained by the latter method could not secrete the fusion protein inthe absence of the p40 subunit and thus, would not be detected by anELISA assay of the culture supernatant. Only cells transfected with bothvectors would secrete the intact fusion protein-p40 heterodimer.

A plasmid vector (pdHL7-14.18-p35) was constructed, as described inGillies, et al., J. Immunol. Methods 125: 191-202 (1989), that containsa dhfr-selectable marker gene, a transcription unit encoding a humanized14.18 anti-GD2 antibody light chain, and a transcription unit encoding ahumanized heavy chain fused to the p35 subunit of human IL-12. Thefusion was achieved by ligation of the XmaI to XhoI fragment of theadapted p35 subunit cDNA, as described in Example 2, to a unique XmaIsite at the end of the CH3 exon of the human IgGI H chain gene. Both theH and L chain transcription units include a cytomegalovirus (CMV)promoter (in place of the metallothionein promoter in the originalreference) at the 5′ end and a poly adenylation site at the 3′ end. Asimilar vector (pC-p40) was constructed for expression of the free p40subunit but did not include a selectable marker gene (dhfr or other) butstill used the CMV promoter for transcription. The coding region in thiscase included the natural leader sequence of the p40 subunit for propertrafficking to the endoplasmic reticulum and assembly with the fusionprotein. Another version of this vector (pNC-p40), which includes theneomycin resistance gene, was constructed for use in sequentialtransfection.

For simultaneous transfection, plasmid DNAs (approximately 10 μg of eachplasmid; pdHL7-14.18-p35 and pC-p40) were linearized by digestion withSalI restriction enzyme, purified using PCR Cleanup kit (Wizard,Promega), and electroporated into 5×10⁶ myeloma cells (in 0.5 ml icecold PBS) using a setting of 0.25 volts and 500 μF. After a recovery for10 min on ice, cells were transferred to fresh medium and plated in96-well dishes at approximately 10⁵ cells/ml. After 48 hr, cells werefed with medium containing methotrexate (0.1 μM). Fresh medium was addedby exchange of half the fluid volume every 4 days until clones appeared.Expression of the desired antibody-IL-12 fusion protein was assayedusing an ELISA based on antibody Fc detection. The capture antibodyreacted with human H and L chains, and the detection utilized anantibody specific for human Fc. Positive clones were expanded inselection medium and the product was purified by binding to and elutionfrom protein A Sepharose as described above. Eluted proteins wereanalyzed by PAGE and detected by staining with Coomassie Blue.

4.2 Sequential Transfection

For sequential transfection, plasmid pNC-p40 was electroporated intocells, as described above, and cells were plated and selected inG418-containing medium. Culture supernatants from drug-resistant cloneswere tested by ELISA for production of p40 subunit. The capture antibodywas a mouse anti-human IL-12 p40 and the detecting antibody was directedto human IL-12 p40/p70. Commercial ELISA kits are available from severalmanufacturers for this purpose (Pharminogen, San Diego; R & D Systems,MN). The highest producing cell clones were tested for the stableexpression of p40. One such clone was transfected with pdHL,7-14.18-p35plasmid DNA, as described above, and clones were selected inmethotrexate-containing medium. Expression of the desired antibody-IL-12fusion protein was assayed using an ELISA based on antibody Fcdetection. The capture antibody reacted with human H and L chains, andthe detection utilized an antibody specific for human Fc. Positiveclones were expanded in selection medium and the product was purified bybinding to and elution from protein A Sepharose as described above.Eluted proteins were analyzed by PAGE and detected by staining withCoomassie Blue.

4.3 Activities of Antibody-IL-12 Fusion Proteins

As summarized in Table 1, fusion protein-expressing cell clones wereobtained by either simultaneous transfection and sequential transfectionbut more highly productive clones were obtained using sequentialtransfection. The product secreted by two individual transfectants wereanalyzed for chain composition. The SDS-PAGE analysis is shown in FIG.5A. Clearly, both clones secrete the same relative amount of each of thethree chains: light chain, H chain-p35, and covalently bound p40,indicating complete and proper assembly of this 6-chain molecule. Thesame process was repeated with a second antibody, KS-¼, reactive withthe EpCAM antigen expressed on virtually all epidermal carcinoma cells(colon, lung, breast, prostate, pancreatic, ovarian, and bladdercarcinoma). Exactly the same results were obtained, including normalbinding activities of the antibodies to their respective antigens.

The biological activities of the whole antibody-IL-12 fusion proteinsare shown in FIG. 5. When assayed for ability to stimulate proliferationof mitogen-activated human PBMC, the Hu-KS-IL-12 fusion protein withboth human IL-12 chains was nearly as active on a molar basis as thehuman IL-12 standard (FIG. 5B). The same construct containing the mousep35 subunit fused to Hu-KS-¼ was significantly less active in thestimulation of human PBMC. When assayed for ability to induce IFN-γsecretion from PHA-activated PBMC, the Hu-KS-IL-12 protein with humanIL-12 chains was about 6-fold less active than the IL-12 standard, whilethe hybrid form was an additional 4-fold less active (FIG. 5C). Whenmouse effector cells (pre-stimulated with Concanavalin A) were used, thehybrid form was about 50-fold less active than the mouse IL-12 standard.The all-human form was inactive (FIG. 5D), as expected from theliterature. See, Schoenhaut, et al., J Immunol. 148: 3433-3340 (1992).

TABLE 1 Comparison of Co-transfection and Sequential Transfection ofIL-12 Fusion Protein Expression Frequency of Method Positive ClonesExpression Level (ng/ml) Co-transfection  4/22 20, 22, 244, 386Sequential 26/37 18, 19, 19, 45, 48, 60, 67, 93, 97, 128, 177, 244, 256,345, 348, 366, 371, 386, 504, 554, 731, 757, 821, 2000

EXAMPLE 5 Expression of Single Chain IL-12 Fusion Proteins

The methods just described for the production of dimeric antibody andFc-based fusion proteins can also be used in its simpler form to expresssingle chain fusion proteins with IL-12 (those not forming dimers). Inthis case, a single polypeptide encoding sequence is joined to thesequence for the p35 subunit and co-expressed in the same cell as thefree p40 subunit. Either of the two methods, simultaneous or sequentialtransfection, can be used to produce single-chain heterodimeric fusionproteins. The purpose of such fusion proteins can be either to targetIL-12 to an antigen bearing cell, through the fusion of a single-chainFv (sc-Fv) antibody (Huston and Oppermann, WO 88/09344) or to combinethe very specific immunostimulatory effect of IL-12 together with aprotein antigen as an adjuvant. The linking of stimulatory protein andantigen ensures their co-localization following injection into ananimal. The antigen can be any polypeptide. These can induce antibodiesin animals capable of reacting with tumor, viral or other antigens thathave therapeutic value. For example, sc-Fv can be used as it is oftenadvantageous to induce immune responses to antibody V regions includingthe idiotype (specific antigen binding region) for the purpose ofstimulating idiotype networks.

The type of antigen used for such fusion proteins can also be one thatnormally induces an allergic response, such as the Der p I and Der p IIfrom dust mites, or tropomyosin from several types of shellfish, whichcan be fused at the DNA level to the p35 subunit of IL-12 and expressedin the same cell with the p40 subunit. Immunization with such fusionproteins would induce strong Th1 helper cell responses that would beuseful in desensitizing the disease-causing Th2 response in atopicpatients with allergy.

To demonstrate the expression of a single chain fusion protein, a scFvversion of the KS-¼ antibody was constructed. The 5′ end of theprotein-encoding portion of fusion gene (an XbaI to AflII fragment)consists of a leader sequence derived from a mouse k light chain, fusedto the mature protein sequence of the KS-¼ L chain V region. The end ofthe V region is fused, in frame, to a DNA encoding the simple linkersequence, (Gly₄Ser)₃, described by others (Huston and Oppermann, WO88/09344) followed, in frame, by the sequence encoding the H chain Vregion of KS-¼. The 3′ end of this scFv contains a XmaI site, compatiblewith ligation to the 5′ end of the human and mouse versions (XmaI toXhoI fragments) of the p35 subunit of IL-12. The final XbaI to XhoIfragments were inserted into the corresponding sites of the sameexpression vector (pdC) used to express the free IL-12 subunits to givevectors pdC-SCA-hu-p35 and pdC-SCA-mu-p35.

These vectors were introduced into a human p40 expressing cell line andgrown in medium containing methotrexate (0.1 μM). Fusionprotein-expressing, drug-resistant clones were identified by ELISAassays specific for the species of p35 utilized in the construct (i.e.,an IL-12 human p40 antibody was used for antigen capture, and specificanti-mouse or human-p35 antibodies were used for detection). Culturemedia from each type of single-chain fusion protein were used todetermine their amounts so that relative specific activities could becalculated. Serial dilutions of each sample were tested for the abilityto induce IFN-γ secretion as detailed above in Example 2. The resultsare shown in FIG. 6, which compares the activity of single-chain IL-12fusion proteins made with either both human subunits or with mouse p35and human p40, as well as the species specificity of the fusionproteins. The data show that the human IL-12 single chain fusion proteinis as active as the whole antibody fusions in its ability to induceIFN-γ but that it is not as potent as the human IL-12 standard whenhuman PBMC were used (FIG. 6A). The hybrid mouse/human form wasapproximately 50-fold less than the mouse IL-12 control as was seen withthe whole antibody construct (FIG. 6B). FIG. 6C shows an antigen bindingassay of the single-chain IL-12 proteins. Plates were coated with the KSantigen recognized by the KS-¼ antibody and used to capture any reactiveantibody or antibody fusion protein. After washing several times, thebound fusion protein was detected using an anti-human IL-12 p40antibody. The data show that the single-chain fusion proteins bound tothe antigen coated plate and could be detected with an antibody againstIL-12, thus demonstrating that the fused molecules retain antigenbinding activity. The intensity of binding was roughly 3-fold lower thanthat seen with the whole KS-¼ antibody but this is not unexpected, dueto the monovalency of the single chain construct.

The activity results with both whole antibody and single chain IL-12fusion proteins suggest that the amino terminus of the p35 chain may beimportant to receptor binding since fusions appear to reduce activity.Nonetheless, the antibody-IL-12 molecules are still very potent inducersof IFN-γ at concentrations above 1 ng/ml. The concentration of suchmolecules in treated animals is expected to be several orders ofmagnitude higher than this both in the circulation, and at the targetsite of action.

A possible way to increase the specific activity of antibody-IL-12fusion proteins would be to insert a flexible peptide linker between theantibody and p35 sequences thus giving more freedom to the aminoterminal sequences of this subunit. A sequence such as the (Gly₄Ser)₃linker, described above, could be used in this manner. One possibleproblem with this approach is that such a linker could be immunogenic,especially when fused to a powerful immune stimulator such as IL-12.

EXAMPLE 6 Pharmacokinetic Properties of IL-12 Fusion Proteins

The antibody-IL-12 fusion proteins were tested for their pharmacokineticbehavior following intravenous injection into Balb/c mice. Blood wascollected from mice by retro-orbital bleeding and stored at 4° C. inEppendorf micro-centrifuge tubes. ELISA methods were used to measure theamount of human antibody, as well as the amount of intact IL-12 fusionprotein, remaining in the blood at increasing time points. The firstELISA measuring human antibody utilizes an antibody against human H andL chains for capture and an anti-human Fc antibody for detection. Thefusion protein-specific assay uses the same first capture step, but ananti-p40 subunit antibody for detection. As depicted in FIG. 7, both theantibody and IL-12 fusion protein had a prolonged half-life but thehalf-life of the fusion protein was somewhat shorter. This suggests thatthe circulating fusion protein is cleaved over time to release IL-12while the antibody remains in the circulation. Earlier-reportedexperiments with other antibody-cytokine fusion proteins demonstratethat cytokines can be released by protease cleavage. See, Gillies, etal., Bioconj. Chem. 4:230-235 (1993). Nonetheless, the half-lives of thefusion proteins are far longer than the 3 hr value reported for nativeIL-12. In fact, the serum concentration at 72 hr is still much higherthan the level required to induce IFN-γ secretion. Trincieri, Blood 84:4008-4027 (1992).

EXAMPLE 7 Treatment of Established Colon Carcinoma with Antibody-IL-12Fusion Protein.

The murine colon carcinoma, CT26, is particularly insensitive totreatment with systemic administration with mouse IL-12 at non-toxicdoses. Martinotti, et al., Eur. J. Immunol. 25: 137-146 (1995). Someefficacy has been found when systemic IL-12 administration has beencombined together with repeated vaccination of irradiated CT26 cells,engineered to secrete IL-2. Vagliani, et al., Cancer Res. 56: 467-470(1996). An alternative approach to successful therapy involved theengineering CT26 to secrete low levels of IL-12. This was ineffectiveunless mice were first treated with antibodies to deplete CD4+ cells,Martinotti, et al., Eur. J. Immunol. 25: 137-146 (1995), presumably dueto an immunosuppressive effect of these cells after exposure to theengineered tumors in vivo. Still another approach of engineering muchhigher IL-12 secretors was far more successful, thus indicating that theamount of local IL-12 was critical in establishing an immune response tosubcutaneous tumors, Colombo, et al., Cancer Res. 56: 2531-2534 (1996).In this case, however, there was no demonstration of treatment ofestablished, disseminated tumors similar to what would be seen in theclinical setting. The purpose of the present experiment was to evaluatethe efficacy of antibody-IL-12 fusion proteins for the treatment ofmurine colon carcinoma, CT26.

CT26 cells were transfected with a cDNA encoding the antigen recognizedby the KS-¼ antibody, referred to as either KS antigen (KSA) orepithelial cell adhesion molecule (EpCAM). Clones expressing thisprotein on their surface were identified by immunostaining with KS-¼ andfluorescence activated cell sorting (FACS) analysis. Cells from oneclone, stably expressing KSA (clone 21.6), were injected into the tailvein of Balb/c mice (1×10⁵ per mouse). Untreated mice formed extensivepulmonary metastases by day 28 and died within 40 days of inoculation.This growth rate was virtually the same as the parental cells indicatingthat the expression of the human KSA had no effect on CT26immunogenicity or ability to form tumors.

The efficacy of the antibody-IL-12 fusion protein for therapy of CT26metastases was tested in this mouse model using the hybrid human/mouseform which has activity on mouse cells. Following tumor cell injection,mice received injections of either PBS (no treatment control), theKS-¼-IL-2 fusion protein (positive control), KS-¼ antibody with freeIL-2 (negative control) or the KS-¼-IL-12 fusion protein (test sample).Treatment began on day 4, a time when established metastases are readilydetectable by histological staining in the lungs of animals, andcontinued daily for 5 days. On day 28 after tumor cell inoculation,animals were euthanized and their lungs examined for the presence oftumor. The weights of the lungs were also measured to determine theamount of tumor mass, relative to tumor-free mice. The results aresummarized in Table 2. Untreated animals had extensive metastaticdisease characterized by near complete surface coverage of the organwith tumor via fusion of individual metastatic nodules. The weights ofthe lungs increased by an average of three-fold, indicating that thetumor masses actually made up the majority of the organ. Treated animalshad little if any evidence of metastases, with some animals completelyfree of tumor. None of the animals showed any overt sign of toxicityduring the treatment process. Thus, unlike treatment with systemicIL-12, antibody-IL-12 fusion protein therapy can eradicate establishedmetastatic CT26 colon carcinoma.

TABLE 2 Treatment of Murine Colon Carcinoma Lung Metastases in SCID Micewith Antibody-IL-12 Fusion Proteins Treatment Metastatic Score OrganWeights PBS 3, 3, 3, 3, 3, 3 0.52 Hu-KS1/4 3, 3, 3, 3, 3 0.48Hu-KS-1/4 + IL-2 3, 3, 3, 3, 3 0.40 Hu-KS-IL-2 2, 1, 1, 1, 1 0.22Hu-KS-IL-12 1, 1, 1, 1, 1 0.20 Experimental lung metastases were inducedby intravenous injection of 10⁵ CT26-KSA cells. Treatment began threedays later with intravenous injection of 10 μg of the humanized KS-1/4antibody or the indicated fusion protein for five consecutive days.Animals were sacrificed and the metastatic score was determined by theextent of surface coverage: 0 = no visible metastatic foci; 1 = lessthan 5% of the surface covered; 2 = 5 to 50% of the surface covered; and# 3 = more than 50% of the lung surface is covered with metastatic foci.

EXAMPLE 8 IL-12 Fusion Proteins as Vaccines.

The humanized KS-¼ antibody IL-12 fusion protein in PBS buffer, madewith the murine p35 subunit (HuKS-¼-mIL-12), was injected into Balb/cmice intravenously (5 μg/day×5). Control mice received the sameantibody, in the same amounts, but with no attached IL-12. Neitherinjection solution contained any other type of adjuvant. On day 10,blood samples were collected into microcentrifuge tubes by retro-orbitalbleeding and plasma were prepared by collecting blood samples in plastictubes containing sodium citrate, followed by centrifugation at fullspeed in an Eppendorf tabletop microcentrifuge. ELISA plates (96-well)were coated with the HuKS-¼ protein containing human constant region andused to capture any mouse antibodies made in response to theimmunization. After washing away unbound material, the bound mouseantibodies were detected with goat anti-mouse Fc antibody (JacksonImmunoResearch) coupled to horse-radish peroxidase. Any bound antibodiescould be directed to either the human constant regions or the variableregion, both of which are shared between the HU-KS-¼ and the fusionproteins.

As depicted in FIG. 8, there was little or no reactivity to Hu-KS-¼without fused IL-12. The fusion protein, on the other hand, induced astrong antibody response in the absence of exogenous adjuvants anddespite the fact that the intravenous route of administration is highlyunfavorable for inducing such responses, compared to either subcutaneousor intraperitoneal administration. Antibodies of the IgG2a isotype,which are typical of IL-12 enhanced responses, were seen in theantibody-IL-12 injected group but not the group injected with theHu-KS-¼ antibody.

The immunogenicity of IL-12 fusion proteins administered by variousroutes is tested by injecting a solution of the fusion protein (such asthat described above) in PBS or other biocompatible buffer, or a knownadjuvant such as Freund's incomplete or complete adjuvant. For example,single or multiple subcutaneous, intradermal or intraperitonealinjections can be given every two weeks. Alternatively, the fusionprotein can be administered first by subcutaneous injection and thenfollowed by intraperitoneal injection. Freund's adjuvant cannot be usedfor human use, due to the irritation at the injection site. Alternativeadjuvants such as precipitates of aluminum hydroxide (Alum) are approvedfor human use and can be used in the present invention. New organicchemical adjuvants based on squalenes and lipids can also be used forinjections into the skin.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A heterodimeric fusion protein comprising a first and a secondchimeric chain, said first chimeric chain comprising a portion of an Igheavy chain linked by a peptide bond to a p35 subunit of interleukin-12(IL-12), said second chimeric chain comprising a portion of an Ig heavychain linked by a peptide bond to a p40 subunit of IL-12, said first andsecond chimeric chains being linked by a disulfide bond.
 2. A fusionprotein comprising a first chimeric Ig chain comprising a portion of anIg heavy chain linked by a peptide bond to a p35 subunit of IL-12, saidp35 subunit of IL-12 being linked to a p40 subunit of IL-12.
 3. Thefusion protein of claim 2 further comprising a second chimeric Ig chaincomprising a portion of an Ig heavy chain linked by a peptide bond to ap35 subunit of IL-12, said p35 subunit of IL-12 being linked to a p40subunit of IL-12, said first and second chimeric chains being linked bya disulfide bond.
 4. A method of increasing the circulating half-life ofIL-12, comprising the steps of: (a) linking a p35 subunit of IL-12 by apeptide bond to a polypeptide, thereby forming a first chimeric chain,said p35 subunit of IL-12 being linked to a p40 subunit of IL-12 by adisulfide bond; (b) linking a p35 subunit of IL-12 by a peptide bond toa polypeptide, thereby forming a second chimeric chain, said p35 subunitof IL-12 being linked to a p40 subunit of IL-12 by a disulfide bond; and(c) linking said first and said second chimeric chain by a disulfidebond, thereby forming a dimeric fusion protein, said fusion proteinhaving a longer circulating half-life than unlinked p35 and p40 subunitsof IL-12.
 5. The method of claim 4 wherein said first and secondpolypeptides are selected from the group consisting of a portion of anIg heavy chain, a portion of an Ig light chain, an antigen, and serumalbumin.
 6. A fusion protein comprising a first chimeric Ig chaincomprising a portion of an Ig heavy chain linked by a peptide bond to ap40 subunit of IL-12, said p40 subunit of IL-12 being linked to a p35subunit of IL-12.
 7. The fusion protein of claim 6 further comprising asecond chimeric Ig chain comprising a portion of an Ig heavy chainlinked by a peptide bond to a p40 subunit of IL-12, said p40 subunit ofIL-12 being linked to a p35 subunit of IL-12, said first and secondchimeric chains being linked by a disulfide bond.
 8. The fusion proteinof claim 6 further comprising a second chimeric Ig chain comprising aportion of an Ig heavy chain linked by a peptide bond to a p35 subunitof IL-12, said p35 subunit of IL-12 being linked to a p40 subunit ofIL-12, said first and second chimeric chains being linked by a disulfidebond.